The contractility of vascular smooth muscle cells (SMC) encircling arteries and arterioles is critical for blood flow regulation. Localized Ca2+ signaling modalities, typified by Ca2+ sparks generated by release of Ca2+ from the sarcoplasmic reticulum (SR) that are functionally coupled with Ca2+-dependent ion channels on the plasma membrane (PM) have significant influence on SMC contractility. Although these indispensable pathways are reliant on tight spatial alignment of the SR with the PM, little is currently known about the structural organization of these membranes in contractile SMC. Thus, the major objective of the research proposed here is to elucidate the molecular architecture and functional significance of peripheral coupling sites maintaining close (?20 nm) interactions between the PM and SR in native SMCs from mouse and human resistance arteries. We will test the original mechanistic hypothesis that junctophilin-2 (JPH2), a protein essential for dyad formation and excitation-contraction coupling in cardiac muscle, and stromal interaction molecule 1 (STIM1), traditionally viewed only as a sensor of Ca2+ store depletion, orchestrate the formation of these sites in native SMCs. We further propose, supported by our unpublished preliminary data, that these structures are critically important for defining the spatial and temporal properties of subcellular Ca2+ signals regulating ion channel activity, membrane potential, and contractility of SMCs and intact arteries. The goal of Aim 1 is to elucidate the molecular architecture of peripheral coupling sites in arterial myocytes. Proposed studies will use a combination of GSDIM super-resolution and live-cell confocal microscopy to test the hypothesis that JPH2 and STIM1 are critically important for SR-PM juxtaposition and the formation of Ca2+ signaling complexes in SMC. The goal of Aim 2 is to elucidate the role of JPH2 and STIM1 in establishing Ca2+ signaling microdomains that regulate Ca2+-activated ion channel activity in arterial myocytes. These studies will use next-generation SMC- specific acta-2?GCaMP5-mCherry Ca2+ biosensor mice, high-speed confocal and TIRFM Ca2+ imaging, and patch-clamp electrophysiology to determine the impact SR-PM interactions maintained by JPH2 and STIM1 on localized Ca2+ signaling and ion channel activity in SMC. Aim 3 will determine the importance of JPH2 and STIM1 in the regulation of arterial contractility. Proposed studies will examine the physiological significance of peripheral coupling sites maintained by JPH2 and STIM1 in loss-of-function experiments investigating fundamental vasomotor responses of intact resistance arteries from mice and human donors. Elucidation of the structure/function relationships of SR-PM complexes in vascular SMC is likely to be highly significant, and will undoubtly shed new insights into vascular function in health and disease, as has been done for cardiac and skeletal muscle. Further, using selective genetic and pharmacological approaches, we will test the hypothesis that disruption of the peripheral coupling architecture impairs regulation of vascular tone, paving the way for future studies aimed at understanding the role of disordered SR-PM interactions in vascular dysfunction.